The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge. The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91. Deleting the valine-88 residue destabilized the protein by 2.93 ± 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 °C. Substitution of alanine for valine-88 stabilized the protein by −0.20 ± 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 ± 0.2 kcal/mol. Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase. The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type. The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism. Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes. The rate constants for refolding transitions were examined by dilution of urea-inactivated protein. While the refolding properties of the V88A mutant were similar to wild type, some rate constants for phases observed in refolding of the valine deletion and the V88I mutant were decreased about 3-fold relative to the wild type. The phase most affected in both these mutants has been previously shown to be related to the formation of the binding site for dihydrofolate during refolding, indicating that the valine-8 8 residue may be at a region that is involved in bringing preformed elements of secondary structure together to form the dihydrofolate binding pocket.